The first, efficient synthesis of novel SLeX neoglycolipids containing N-deacetylated and lactamized sialic acid: Key ligand structures for selectin binding

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THE FIRST, EFFICIENT SYNTHESIS OF NOVEL SLex
NEOGLYCOLIPIDS CONTAINING N-DEACETYLATED AND
LACTAMIZED SIALIC ACID: KEY LIGAND STRUCTURES
FOR SELECTIN BINDING
Nobumasa Otsubo a , Masanori Yamaguchi a , Hideharu Ishida a & Makoto Kiso a
a Department of Applied Bioorganic Chemistry , Gifu University , Gifu, 501-1193, Japan
Published online: 16 Aug 2006.
To cite this article: Nobumasa Otsubo , Masanori Yamaguchi , Hideharu Ishida & Makoto Kiso (2001) THE FIRST, EFFICIENT
SYNTHESIS OF NOVEL SLex NEOGLYCOLIPIDS CONTAINING N-DEACETYLATED AND LACTAMIZED SIALIC ACID: KEY LIGAND
STRUCTURES FOR SELECTIN BINDING , Journal of Carbohydrate Chemistry, 20:3-4, 329-334, DOI: 10.1081/CAR-100104868
To link to this article: http://dx.doi.org/10.1081/CAR-100104868
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J. CARBOHYDRATE CHEMISTRY, 20(3&4), 329–334 (2001)
COMMUNICATION
THE FIRST, EFFICIENT SYNTHESIS OF NOVEL
SLex NEOGLYCOLIPIDS CONTAINING
N-DEACETYLATED AND LACTAMIZED SIALIC
ACID: KEY LIGAND STRUCTURES FOR
SELECTIN BINDING1
Nobumasa Otsubo, Masanori Yamaguchi, Hideharu Ishida, and
Makoto Kiso
Department of Applied Bioorganic Chemistry, Gifu University,
Gifu 501-1193, Japan
Sialyl Lewis x (sLex) has been recognized2 as a common carbohydrate ligand for E-,
P- and L-selectin, a family of C-type lectins implicated in lymphocyte homing, leuko-
cyte recruitment to sites of inflammation, thrombosis, cancer metastasis, and so on.
Recently, it has been suggested3,4 that the novel sLex variants containing N-deacety-
lated and lactamized sialic acid may be involved in the ligand processing pathway for
human L-selectin, raising a new regulation mechanism of ligand activity based on the
heterogeneity of sialic acid in the sLex determinant (Figure 1). This paper reports the
first, efficient synthesis of novel sLex neoglycolipids which contain N-deacetylated
and lactamized sialic acid as the key ligand structures for selectin binding.
Phenyl 4,6-O-benzylidene-2-deoxy-3-O-(4-methoxybenzyl)-2-phthalimido-
1-thio-ꢀ-D-glucopyranoside5 (1, 1.71 mmol) was coupled with 2 (1.23 mmol) which
was readily prepared from 2-(trimethylsilyl)ethyl 3-O-benzyl-ꢀ-D-galactopyra-
noside,6 in the presence of N-iodosuccinimide (NIS, 3.37 mmol), trifluo-
romethanesulfonic acid (TfOH, 0.17 mmol) and molecular sieves 4Å (MS 4A, 2.0
g) in CH2Cl2 at ꢁ20°C, to give 3, [ꢂ]D ꢃ21° (CHCl3), in 92% yield (Scheme 1).
Treatment of 3 with hydrazine monohydrate in EtOH for 24 h under reflux, followed
by successive N-acetylation and O-benzoylation, gave 4, [ꢂ]D ꢃ31° (CHCl3), in
91% yield. The benzylidene group in 4 was cleaved by acid hydrolysis, and the re-
sulting 5 was treated with p-methoxyphenol (MPOH), PPh3 and diethylazodicar-
boxylate (DEAD) in THF to afford 6, [ꢂ]D ꢃ28° (CHCl3), in 96% yield.
Glycosylation of 6 (0.37 mmol) with the suitably protected N-trifluo-
roacetylneuraminyl-ꢂ-(2→3)-galactose donor 7 (0.46 mmol), which was prepared
329
Copyright © 2001 by Marcel Dekker, Inc.
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OTSUBO ET AL.
Figure 1. Hypothetical ligand-processing pathway for human L-selectin.3,4
from the corresponding trichloroacetimidate derivative by the similar manner re-
ported previously,3 promoted by dimethyl(methylthio)sulfonium triflate7
(DMTST, 1.85 mmol) and MS 4A (1.0 g) in CH2Cl2 at 0°C, gave 8, [ꢂ]D ꢃ38°
(CHCl3), in 75% yield. In the 1H NMR spectrum of 8, a significant one-proton dou-
blet (J1,2 ꢄ 8.0 Hz, H-1c) appeared at ꢅ 5.06, showing the newly formed glycosidic
linkage to be ꢀ. The p-methoxybenzyl (MPM) group at C-3 of GlcNAc in 8 was
selectively removed (82%) by treatment with TMSCl, SnCl2 and anisole in CH2Cl2
at 0°C, and the resulting 9 (0.23 mmol) was fucosylated by 10 (0.71 mmol) in the
presence of NIS (2.1 mmol) and TfOH (0.56 mmol) in benzene at 7°C to afford the
desired pentasaccharide 11, [ꢂ]D ꢃ3.0° (CHCl3), in 85% yield. Hydrogenolytic re-
moval of the benzyl groups in the fucose moiety and the following O-acetylation
gave 12 (Scheme 1) in 81% yield. In the 1H NMR spectrum of 12, a three-proton
doublet at ꢅ 0.76 (J5,6 ꢄ 6.4 Hz, H-6e), a one-proton doublet of doublets at ꢅ 4.90
(J1,2 ꢄ 3.6, J2,3 ꢄ 9.9 Hz, H-2e), and a one-proton doublet at ꢅ 5.16 (J1,2 ꢄ 3.6 Hz,
H-1e) were clearly detected, indicating the newly formed glycoside to be an ꢂ-L-
fucopyranoside. The pentasaccharide 12 was then converted to the imidate deriva-
tive 13 (ꢂ:ꢀ ꢄ 5:1) by the removal of SE group (quant.) and activation as the
trichloroacetimidate in 89% yield.
Coupling of 13 (0.09 mmol) and 2-(tetradecyl)hexadecanol8 14 (0.3 mmol) in
the presence of trimethylsilyl trifluoromethanesulfonate (TMSOTf, 8.02 ꢆmmol)
and molecular sieves AW-300 in CH2Cl2 gave the desired neoglycolipid 15, [ꢂ]D
ꢃ8.4° (CHCl3), in 70% yield (Scheme 2). Significant signals in the 1H NMR spec-
trum of 15 were a six-proton triplet at ꢅ 0.88 (Jvic ꢄ 6.0 Hz, 2Me), fifty-three alkyl
protons at ꢅ 0.93–1.52 (26CH2 and CH) and a one-proton doublet at ꢅ 4.33

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KEY LIGAND STRUCTURES FOR SELECTIN BINDING
331
Scheme 1. MPMꢄp-methoxybenzyl, SEꢄ2-(trimethylsilyl)ethyl, MPꢄp-methoxyphenyl,
TFAcꢄtrifluoroacetyl.
(J1,2 ꢄ 8.0 Hz, H-1a), characteristic of the desired ꢀ-linked 2-(tetradecyl)hexade-
cyl glycoside. The MP group was selectively cleaved by treatment with diammo-
nium cerium(IV) nitrate (CAN) at ꢁ10°C in CH3CN-H2O to give 16, [ꢂ]D ꢁ20°
(CHCl3) in 85%. Removal of the O-acyl and N-trifluoroacetyl groups with NaOMe

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OTSUBO ET AL.
Scheme 2.

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KEY LIGAND STRUCTURES FOR SELECTIN BINDING
333
in MeOH, and subsequent saponification of the methyl ester group by addition of
water afforded the desired N-deacetylated sLex neoglycolipid 17, [ꢂ]D ꢁ19° (3:1
MeOH-CHCl3), in 93% yield.
Treatment of 17 (10.3 ꢆmol) with 1-(3-dimethylaminopropyl)-3-ethylcar-
bodiimide hydrochloride (WSC, 0.1 mmol) in dimethyl sulfoxide (DMSO, 2 mL)
for 10 h at 60°C gave the desired lactamized sLex 18, [ꢂ]D ꢁ16.3° (3:2 CHCl3-
MeOH), in 75% yield. In the 1H NMR spectra (500 MHz) of 17 and 18 in CD3OD,
H-3 of the N-deacetylated sialic acid moiety appeared at ꢅ 1.73 as a one-proton
triplet (Jgem ꢄ J3,4 ꢄ 12.6 Hz, H-3dax), and at ꢅ 2.86 as a one-proton doublet of
doublets (J3eq,4 ꢄ 4.2 Hz, H-3deq), respectively, showing the usual 2C5 chair con-
formation. In contrast, H-3 of the lactamized sialic acid moiety in 18 appeared at ꢅ
2.03 (Jgem ꢄ 13.9, J3ꢂ,4 ꢄ 4.8 Hz, H-3dꢂ) and ꢅ 2.29 (Jgem ꢄ 13.9, J3ꢀ,4 ꢄ 10.6 Hz,
H-3dꢀ), respectively, as a one-proton doublet of doublets, obviously indicating a
typical B5,2 boat conformation. These 1H NMR data are consistent with those re-
ported9 for the ganglioside GM4 analogs containing N-deacetylated and lac-
tamized sialic acid.
In conclusion, an efficient synthesis of the novel sLex neoglycolipids con-
taining N-deacetylated and lactamized sialic acid was achieved for the first time.
ACKNOWLEDGMENTS
This work was supported in part by Grants-in-Aid (No. 12306007 and No.
12045228) for Scientific Research from the Ministry of Education, Science and
Culture of Japan, and Japan Society for Promotion of Science.
REFERENCES AND NOTES
1. Synthetic Studies on Sialoglycoconjugates, Part 120. For Part 119, see Sawada, N.; Ito,
M.; Ishida, H.; Kiso, M. The First Synthesis of Glycan Parts of Lactoganglio- and Ne-
olactoganglio-Series Gangliosides. Tetrahedron Lett. 2001, in press.
2. Simanek, E. E.; McGarver, G. J.; Jablonowski, J. A.; Wong, C.-H. Selectin-carbohy-
drate Interactions: from Natural Ligands to Designed Mimics. Chem. Rev. 1998, 98,
833–862.
3. Komba, S.; Galustian, C.; Ishida, H.; Feizi, T.; Kannagi, R.; Kiso, M. The First Total
Synthesis of 6-Sulfo-de-N-acetylsialyl Lewis x Ganglioside: A Superior Ligand for
Human L-Selectin. Angew. Chem. Int. Ed. 1999, 38(8), 1131–1133.
4. Mitsuoka, C.; Ohmori, K.; Kimura, N.; Kanamori, A.; Komba, S.; Ishida, H.; Kiso, M.;
Kannagi, R. Regulation of Selectin Binding Activity by Cyclization of Sialic Acid
Moiety of Carbohydrate Ligands on Human Leukocytes. Proc. Natl. Acad. Sci. USA
1999, 96, 1597–1602.
5. Kameyama, A.; Ehara, T.; Yamada, Y.; Ishida, H.; Kiso, M.; Hasegawa, A. A Total
Synthesis of Sialyl Dimeric Lex Ganglioside. J. Carbohydr. Chem. 1995, 14, 507–523.
6. Hasegawa, A.; Hotta, K.; Kameyama, A.; Ishida, H.; Kiso, M. Total Synthesis of Sia-
lyl-ꢂ(2-6)-lactotetraosylceramide and Sialyl-ꢂ(2-6)-neolactotetraosylceramide. J.
Carbohydr. Chem. 1991, 10, 439–459.

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OTSUBO ET AL.
7. Kiso, M.; Hasegawa, A. Synthesis of Ganglioside GM3 and Analogs Containing Mod-
ified Sialic Acids and Ceramides. In Methods in Enzymology; Abelson, J. N.; Simon,
M. I. Eds.; Academic Press.; San Diego, 1994, 242, 173–182.
8. Hasegawa, A.; Suzuki, N.; Ishida, H.; Kiso, M. Synthesis of Ganglioside GM3 and
GM4 Analogs Containing 2- or 3-Branched Fatty-Alkyl Residues in Place of Ce-
ramide. J. Carbohydr. Chem. 1996, 15(5), 623–637.
9. Otsubo, N.; Ishida, H.; Kiso, M. Synthesis of Novel Ganglioside GM4 Analogues Con-
taining N-Deacetylated and Lactamized Sialic Acid: Probes for Searching New Ligand
1
Structures for Human L-Selectin. Carbohydr. Res. 2001, 330(1), 1–5. The H NMR
data (500 MHz, CD3OD) for N-deacetylated and lactamized sialic acid in the GM4
analogues are as follows: ꢅ 1.71 (t, J ꢄ 12.1 Hz, H-3ax) and ꢅ 2.87 (dd, Jgem ꢄ 12.5,
J
3,4 ꢄ 4.8 Hz, H-3eq) for the N-deacetylated sialic acid; ꢅ 2.02 (dd, Jgem ꢄ 13.9, J3ꢂ,4
ꢄ 4.8 Hz, H-3ꢂ) and ꢅ 2.29 (dd, Jgem ꢄ 13.9, J3ꢀ,4 ꢄ 10.3 Hz, H-3ꢀ) for the lactamized
sialic acid. The FT-IR (KBr) spectrum showed a significant absorption band at 1690
cmꢁ1 (lactam).
Received December 25, 2000
Accepted February 5, 2001

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